We have examined creatine uptake by confluent monolayers of IMR-90 fibroblasts, smooth muscle cell strains derived from calf aorta and human uterus, and a rat skeletal muscle cell line. All of these cell types had the ability to concentrate creatine in the culture medium. The kinetics of uptake suggest that the process is mediated, is dependent on the presence of Na ion in the medium, and is heterogeneous. We propose to characterize the process of translocation of creatine across the plasma membrane. Initial rates of uptake of creatine by intact cells, and by plasma membrane vesicles prepared from them, will be measured by rapid sampling techniques. Na ion dependent and Na ion independent components of transport will be characterized with respect to kinetic behavior and its modification by metabolic inhibitors and structural analogs of creatine. Initial rates of uptake of creatine, intracellular concentrations of creatine phosphate in various steady states, and rates of efflux of creatine will be compared in the various cell types. Phosphorylation of creatine taken up by the cells results in large increases in the size of the intracellular phosphagen pool. The effect of the expanded phosphagen pool on steady state levels of ATP and ADP and on the regulation of respiration and glycolysis will be examined in different types of resting cells and in cell subjected to electrical or mechanical stimulation.